Relationship between gyrA gene mutation in Mycobacterium tuberculosis and the minimum inhibitory concentration of fluoroquinolones in vitro
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摘要:目的 探讨结核分枝杆菌gyrA基因突变与氟喹诺酮类药物体外最低抑菌浓度（MIC）的关系。方法 收集640例痰结核分枝杆菌阳性临床分离株，用探针熔解曲线法筛选gyrA基因耐药决定区突变株，对其gyrA基因耐药决定区进行测序，并检测氧氟沙星、左氧氟沙星、莫西沙星及加替沙星对不同突变位点菌株的MIC，分析其差异。结果 探针熔解曲线法筛选出gyrA突变株共45株（7.03%），其中90位点突变15株（33.33%）、94位点突变26株（57.78%）、91位点突变4株（8.89%）。90位点突变菌株中，氧氟沙星高水平耐药2株，左氧氟沙星无耐药，莫西沙星低水平耐药3株、高水平耐药2株，加替沙星高水平耐药3株；91位点突变菌株中，氧氟沙星、左氧氟沙星无耐药，莫西沙星高水平耐药2株，加替沙星高水平耐药1株；94位点突变菌株中，氧氟沙星低水平耐药4株、高水平耐药7株，左氧氟沙星低水平耐药7株，莫西沙星低水平耐药2株、高水平耐药13株，加替沙星低水平耐药3株、高水平耐药14株。除左氧氟沙星外，90位点突变菌株对其他3种药物易出现高水平耐药；91位点突变菌株对莫西沙星易出现高水平耐药；94位点突变菌株对莫西沙星、加替沙星多为高水平耐药，其中天冬氨酸（Asp）→天冬酰胺（Asn）和Asp→甘氨酸（Gly）突变类型的菌株更易出现。结论 结核分枝杆菌gyrA基因突变类型与氟喹诺酮类药物MIC相关，可为临床用药提供重要参考。Abstract:Objective To explore the relationship between gyrA gene mutation in Mycobacterium tuberculosis and the minimum inhibitory concentration (MIC) of fluoroquinolones in vitro.Methods Clinical isolated positive strains of Mycobacterium tuberculosis in 640 patients were collected. The probe solubility curve was used to screen strains with mutation of gyrA gene in the drug resistance determining region, and sequencing of the drug resistance determining region was performed. The MICs of ofloxacin, levofloxacin, moxifloxacin and gatifloxacin for strains with mutations at different loci were detected, and the differences were analyzed.Results A total of 45 strains (7.03%) with gyrA gene mutation were screened out by the probe solubility curve, including 15(33.33%) with the codon 90 mutation, 26(57.78%) with the codon 94 mutation, and 4(8.89%) with the codon 91 mutation. Among strains with the codon 90 mutation, 2 strains showed high resistance to ofloxacin, no levofloxacin-resistant strains were found, 3 showed low resistance to moxifloxacin, 2 showed high resistance to moxifloxacin, and 3 showed high resistance to gatifloxacin. Among strains with the codon 91 mutation, no ofloxacin or levofloxacin-resistant strains were found.Two strains showed high resistance to moxifloxacin, and 1 showed high resistance to gatifloxacin. Among strains with the codon 94 mutation, 4 strains showed low resistance and 7 showed highly resistant to ofloxacin, 7 showed low resistance to levofloxacin, 2 showed low resistance and 13 showed high resistance to moxifloxacin, 3 showed low resistance and 14 showed high resistance to gatifloxacin.For strains with the codon 90 mutation, except for levofloxacin, they were all highly resistant to the other drugs.Strains with the codon 91 mutation were prone to have high resistance to moxifloxacin, and strains with the codon 94 mutation were mostly highly resistant to moxifloxacin and gatifloxacin, especially those with Aspartic acid (Asp)→Asparagine (Asn) and Asp→Glycine (Gly).Conclusion The type of gyrA gene mutation in Mycobacterium tuberculosis is related to the MICs of fluoroquinolones, which provides important reference for clinical medication.
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表 1 结核分枝杆菌分离株gyrA基因突变情况分析
位点 突变类型 氨基酸变化 菌株数/株 占比/% 90 gCg→gTg Ala→Val 15 33.33 91 Tcg→Ccg Ser→Pro 4 8.89 94 Gac→Tac Asp→Tyr 4 8.89 Gac→Aac Asp→Asn 6 13.33 gAc→gCc Asp→Ala 6 13.33 gAc→gGc Asp→Gly 10 22.22 Ala: 丙氨酸; Val: 缬氨酸; Ser: 丝氨酸; Pro: 脯氨酸; Asp: 天冬氨酸; Tyr: 酪氨酸; Asn: 天冬酰胺; Gly: 甘氨酸。 
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表 2 不同类型gyrA基因突变菌株的氧氟沙星耐药情况[n(%)]
突变类型 菌株数/株 表型DST 氧氟沙星MIC/(μg/mL) 低水平耐药菌株 高水平耐药菌株 1.000 2.000 4.000 8.000 16.000 32.000 90Ala→Val 15 0 2(13.33) 4(26.67) 9(60.00) 0 2(13.33) 0 0 91Ser→Pro 4 0 0 0 4(100.00) 0 0 0 0 94Asp→Tyr 4 0 1(25.00) 0 3(75.00) 0 1(25.00) 0 0 94Asp→Asn 6 2(33.33) 2(33.33) 0 2(33.33) 2(33.33) 2(33.33) 0 0 94Asp→Ala 6 0 0 0 6(100.00) 0 0 0 0 94Asp→Gly 10 2(20.00) 4(40.00) 1(10.00) 3(30.00) 2(20.00) 2(20.00) 2(20.00) 0 Ala: 丙氨酸; Val: 缬氨酸; Ser: 丝氨酸; Pro: 脯氨酸; Asp: 天冬氨酸; Tyr: 酪氨酸; Asn: 天冬酰胺; Gly: 甘氨酸; MIC: 最低抑菌浓度。
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表 3 不同类型gyrA基因突变菌株的左氧氟沙星耐药情况[n(%)]
突变类型 菌株数/株 表型DST 左氧氟沙星MIC/(μg/mL) 低水平耐药菌株 高水平耐药菌株 0.250 0.500 1.000 2.000 4.000 8.000 16.000 90Ala→Val 15 0 0 0 8(53.33) 5(33.33) 2(13.33) 0 0 0 91Ser→Pro 4 0 0 0 0 4(100.00) 0 0 0 0 94Asp→Tyr 4 2(50.00) 0 0 0 0 2(50.00) 2(50.00) 0 0 94Asp→Asn 6 3(50.00) 0 0 0 2(33.33) 1(16.67) 3(50.00) 0 0 94Asp→Ala 6 0 0 0 0 2(33.33) 4(66.67) 0 0 0 94Asp→Gly 10 2(20.00) 0 0 0 4(40.00) 4(50.00) 2(20.00) 0 0 Ala: 丙氨酸; Val: 缬氨酸; Ser: 丝氨酸; Pro: 脯氨酸; Asp: 天冬氨酸; Tyr: 酪氨酸; Asn: 天冬酰胺; Gly: 甘氨酸; MIC: 最低抑菌浓度。
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表 4 不同类型gyrA基因突变菌株的莫西沙星耐药情况[n(%)]
突变类型 菌株数/株 表型DST 莫西沙星MIC/(μg/mL) 低水平耐药菌株 高水平耐药菌株 0.250 0.500 1.000 2.000 4.000 8.000 16.000 90Ala→Val 15 3(20.00) 2(13.33) 6(40.00) 4(26.67) 3(20.00) 0 2(13.33) 0 0 91Ser→Pro 4 0 2(50.00) 0 2(50.00) 0 0 2(50.00) 0 0 94Asp→Tyr 4 0 2(50.00) 0 2(50.00) 0 0 2(50.00) 0 0 94Asp→Asn 6 0 4(66.67) 0 2(33.33) 0 0 4(66.66) 0 0 94Asp→Ala 6 2(33.33) 2(33.33) 0 2(33.33) 2(33.33) 0 2(33.33) 0 0 94Asp→Gly 10 0 5(50.00) 0 5(50.00) 0 0 2(20.00) 3(30.00) 0 Ala: 丙氨酸; Val: 缬氨酸; Ser: 丝氨酸; Pro: 脯氨酸; Asp: 天冬氨酸; Tyr: 酪氨酸; Asn: 天冬酰胺; Gly: 甘氨酸; MIC: 最低抑菌浓度。
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表 5 不同类型gyrA基因突变菌株的加替沙星耐药情况[n(%)]
突变类型 菌株数/株 表型DST 加替沙星MIC/(μg/mL) 低水平耐药菌株 高水平耐药菌株 0.125 0.250 0.500 1.000 2.000 4.000 8.000 16.000 90Ala→Val 15 0 3(20.00) 7(46.67) 1(6.67) 3(20.00) 1(6.67) 0 3(20.00) 0 0 91Ser→Pro 4 0 1(25.00) 1(25.00) 1(25.00) 0 1(25.00) 0 1(25.00) 0 0 94Asp→Tyr 4 1(25.00) 2(50.00) 0 1(25.00) 0 0 1(25.00) 2(50.00) 0 0 94Asp→Asn 6 0 4(66.67) 1(16.67) 1(16.67) 0 0 0 4(66.67) 0 0 94Asp→Ala 6 1(16.67) 2(33.33) 0 2(33.33) 0 1(16.67) 1(16.67) 2(33.33) 0 0 94Asp→Gly 10 1(10.00) 6(60.00) 2(20.00) 0 0 1(10.00) 1(10.00) 4(40.00) 2(20.00) 0 Ala: 丙氨酸; Val: 缬氨酸; Ser: 丝氨酸; Pro: 脯氨酸; Asp: 天冬氨酸; Tyr: 酪氨酸; Asn: 天冬酰胺; Gly: 甘氨酸; MIC: 最低抑菌浓度。
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